We plan to isolate supranucleosomal units of chromatin structure from nuclei of Drosophila melanogaster tissue culture cells by mild nuclease digestions followed by sucrose gradient centrifugation of the particles. The isolated supranucleosomal particles will be characterized by physicochemical techniques (viscometry, sedimentation) and electron microscopy. The particles will be redigested with various nucleases to determine the path of the DNA. The proteins will be analysed in SDS and acid urea gels to determine the composition and stoichiometry of the histone and non-histone proteins. In particular, we shall determine the location and role of histone Hl in the nucleosomal clusters using crosslinking agents and limited proteolysis followed by gel electrophoresis analysis of the crosslinking agents and limited proteolysis followed by gel electrophoresis analysis of the crosslinked, cleaved products. We plan to fractionate the supranucleosomal structures in order to separate a) the active (transcribing) from the inactive (heterochromatic) nucleosomal clusters and b) the stationary phase, S phase and M phase supranucleosomal arrays. These various particles will be then characterized as to their protein composition and DNA sequence (using as probes nick translated E. coli plasmids containing known Drosophila sequences). We shall further characterize the chromomere-size DNA loops in interphase and metaphase chromosomes and determine their DNA sequence organization by restriction cleavage, partition of the free (soluble) and bound (nuclear attached) DNA fragments followed by gel electrophoresis and Cot analysis of the fractionated restriction fragments. We shall identify and determine the function of the non-histone proteins and RNA chains associated with the folded interphase genomes.